Over two-thirds of the world's population live in regions where rabies is endemic, resulting in over 15 million people receiving multi-dose post-exposure prophylaxis (PEP) and over 75,000 deaths per year globally. A major goal in rabies virus (RV) research is to develop a single-dose post-exposure prophylaxis (PEP) that would simplify vaccination protocols, reduce costs associated with RV prevention, and save lives. Live replication-deficient RV-based vectors are emerging as promising vaccines to achieve this goal. Multiple signals lead to optimal B cell activation and functions. Among these signals, BAFF and APRIL bridge the gap between innate and adaptive immunity by influencing B and T cell functions. We hypothesize that live replication-deficient RV-based vaccines expressing BAFF or APRIL will induce rapid and robust RV-specific immunity and protection against pathogenic RV challenge more effectively than a parental live RV-based vaccine. The major goal of this application is to investigate B cell activation and RV-specific immunity and protection against pathogenic RV challenge elicited by live replication-deficient RV-based vaccines expressing BAFF or APRIL and to confirm the absence of immune pathology. The goal for the first Aim is to construct, recover and characterize live matrix (M) gene-deleted RV-based vaccine vectors (rRV- M) expressing BAFF or APRIL. The goal of the second Aim is to complete a comprehensive evaluation of the functional consequences of expressing BAFF or APRIL from live RV-based vaccine vectors in the context of RV-specific B cell activation, immunity, protection and safety. Qualitative and quantitative measures of B cell and antibody attributes (B cell activation, virus neutralization activity, antibody avidity and subclass profile), and CD4 T cells (Th1/Th2 bias by intracellular cytokine staining), which are the primary anti-viral responses critical for RV PEP, will be analyzed using a mouse model of RV immunogenicity and protection. The absence of autoimmunity, B cell lymphocytic disorders and extreme inflammatory responses will be monitored to help confirm the new vaccines remain safe. This exploratory grant will potentially identify a single-dose vaccine strategy that exploits the speed by which innate immunity promotes adaptive B cell responses, resulting in antibodies capable of rapidly neutralizing and preventing RV infections. Future goals include additional testing to identify the most effective vaccination strategy, safety, and toxicity in pre-clinical settings with the ultimat goal of reducing the cost of rabies prevention in humans and saving lives.